Apoptosis is cell death resulting from cells' own active induction under physiological conditions. Apoptosis is clearly distinguished from cell death (necrosis) resulting from environmental deterioration. Apoptosis is morphologically characterized by chromosome aggregation in cell nuclei, fragmentation of cell nuclei, disappearance of microvillus on the cell surface layers, aggregation of cytoplasm, and the like. When cells initiate apoptosis, they become atrophied. Intracellular contents are immediately incorporated by macrophages and surrounding cells without being released outside the cells. Thus, inflammation is not induced and the surrounding cells are not affected by apoptosis. Hence, many attempts have been made to treat disease by inducing apoptosis in cells (e.g., cancer cells), the existence of which is harmful to their host organisms.
As means or factors for inducing apoptosis, glucocorticoid treatment, cell damage due to cytotoxic-T cells, atrophy of hormone-dependent tissues, radiation exposure, NK cells, killer cells, tumor necrosis factors (TNF), cytokines such as lymphotoxin (LT), and the like have been reported (Wyllie, A. H., Nature 284: 555-556, 1986; Wyllie, A. H. et al., Int. Rev. Cytol. 68: 251, 1980; Duvall, E. and Wyllie, A. H., Immunology Today, 7: 115-119, 1986; Sellins, K. S. et al., J. Immunol. 139: 3199, 1987; Yamada, T. et al., Int. J. Radiat. Biol. 53: 65, 1988; Schmid, D. S. et al., Proc. Natl. Acad. Sci. USA, 83: 1881-1885, 1986; John, C. et al., J. Immunol. 129 (4): 1782-1787, 1982; Howell, D. M. et al., J. Immunol. 140: 689-692, 1988; Gillian, B. et al., Eur. J. Immunol. 17: 689-693, 1987). Furthermore, it is also known that apoptosis is also induced by some types of antibody (e.g., anti-CD3 antibody and anti-APO-I antibody) (Trauth, B. C. et al., Science 245: 301-305, 1989; Smith, C. A. et al., Nature 337: 181-184, 1989; Tadakuma, T. et al., Eur. J, Immunol. 20: 779, 1990). Moreover, it has also been reported that cycloheximide, a protein synthesis inhibitor, induces apoptosis in acute leukemia cells, that actinomycin D, an RNA synthesis inhibitor, induces apoptosis in small intestine crypt cells, and that both inhibitors induce apoptosis in HL-60 cells (Martin, S. J. et al., J. Immunol. 145: 1859-1867, 1990).
As apoptosis-related therapeutic methods, in addition to the above attempts to treat cancer using an anti-Apo-I antibody, administration of etoposide or aclarubicin against osteomyelodysplasia syndrome (MDS) due to active proliferation of blast cells has been examined (Shibuya, T., J. Clinical and Experimental Medicine 160 (5): 319-323, 1992). In addition to these attempts, inventions relating to methods for inducing apoptosis or drug compositions therefor are known (e.g., JP Patent Publication (Kokai) No. 2001-275681 A; JP Patent Publication (Kohyo) No. 2002-526109 A; JP Patent Publication (Kohyo) No. 10-508575 A; JP Patent Publication (Kokai) No. 9-328425 A; and International Patent Publication WO95/28154 Pamphlet).
In addition, c-Myc protein encoded by the c-myc gene is not only extremely important in cell life activities such as cell proliferation, cell differentiation, and the cell cycle, but is also deeply involved in cellular tumorigensis (transformation). Enhanced c-Myc protein expression is observed in many cancer tissues and in cellular tumorigensis due to the activation of the c-myc gene. c-Myc protein also relates to apoptosis. Both increases and decreases in intracellular expression level of c-Myc protein induce apoptosis (Thompson, E. B. Ann. Rev. Physiol. 60: 575-600, 1998). For example, the suppression of the c-myc gene was found to be essential for apoptosis induction in experiments using glucocorticoid in human leukemia cells (Thulasi, R. et al., J. Biol. Chem. 268: 18306-18312, 1993; Zhou, F. et al., J. Steroid Biochem. Mol. Biol. 73:195-202, 2000; Thompson, E. A. et al., Cancer Res., 51: 5544-5550, 1991; Helmberg, A. et al., EMBO J., 14: 452-60, 1995). In systems using B cells, all chemical substances that induce apoptosis are deeply associated with the suppression of c-myc gene expression (McCormack, J. E. et al., Proc. Natl. Acad. Sci. U.S.A. 81:5546-5550, 1984; Sonenshein, G. E., J. Immunol. 158: 1994-1997, 1997; Fischer, G. et al., J. Exp. Med., 179: 221-228, 1994; Wu, M. et al., Mol. Cell. Biol. 16: 5015-5025, 1996). Furthermore, apoptosis is induced by introduction of an antisense oligonucleotide of c-myc into several types of cell (Thompson, E. B., Ann. Rev. Physiol. 60: 575-600, 1998). In addition, apoptosis is induced by depletion of IL-3 in IL-3-dependent myeloma cells and simultaneous forced expression of the c-myc gene (Askew, D. S. et al., Oncogene 6: 1915-1922, 1991). Moreover, apoptosis is induced by forced expression of the c-myc gene in Rat1 fibroblasts in serum-free media (Evan, G. I. et al., Cell 69: 119-128, 1992).
Such c-Myc protein is produced by transcription of the c-myc gene. The c-myc gene is tightly controlled by many transcription factors, but how transcription is controlled is mostly unknown. For example, an abnormal APC (adenomatous polyposis coli) gene is observed in 70% to 80% of colorectal cancer cases. It is said that such abnormality is developed at the most initial stage of oncogenesis. APC protein binds to β-catenin that is stabilized by the Wnt/Wingless signal transduction pathway, thereby suppressing the functions thereof. β-catenin binds to the Tcf/Lef transcription factor, thereby activating c-myc gene transcription. Hence, it is considered that when abnormalities take place in an APC gene, it becomes impossible to suppress β-catenin activities and the c-myc gene is sustainably activated, thereby inducing cell proliferation.
c-Myc protein expression is under influence of many transcription factors, in addition to the Wnt/Wingless signal transduction pathway. For example, it is known that differentiation of HL60 (human promyeloid leukemia cell) is induced by various chemical substances including DMSO (Dimethyl sulfoxide; Me2SO), retinoic acid, phorbol esters, vitamin D derivatives, and the like. At the time of differentiation, it is known that intracellular c-Myc protein expression is attenuated. These facts suggest that various differentiation-inducing substances activate various transcription factors so as to affect the c-myc gene and that such activations are finally integrated into a single pathway so as to suppress c-myc gene transcription.
Based on such understandings, analysis has been carried out regarding which site located upstream of the c-myc gene affects the transcription. As a result, it has been shown that a site of a hundred and several tens of nucleotides, which is located as far as 1.5 kb upstream of the transcription initiation site of the c-myc gene, is extremely important in c-myc gene transcription. The site has been named FUSE (Far Upstream Element) (Avigan, M. et al., J. Biol. Chem., 265: 18538-18545, 1990). Next, protein that binds to FUSE has been analyzed by oligonucleotide affinity chromatography. Thus, FBP (FUSE-binding protein) having a molecular weight of 70 kDa has been identified. Furthermore, the FBP protein has its own strong transcriptional activity. It is shown that the FBP protein may control the c-myc gene (Bazar, L. et al., J. Biol. Chem., 270: 8241-8248, 1995; Duncan, R. et al., Genes Dev., 8:465-480, 1994; Michelotti, G. A. et al., Mol. Cell. Biol. 16:2656-2669, 1996). Moreover, FIR (FBP Interacting Repressor) has been identified as protein that binds to (interacts with) the FBP protein (Liu, J. et al., Mol. Cell, 5: 331-341, 2000). Such FIR has been shown to suppress c-myc gene transcription by suppressing the functions of a basic transcription factor TFIIH (Liu, J. et al., Cell, 104: 353-363, 2001). However, FIR has never been known to induce apoptosis.
As described above, c-Myc protein is deeply involved in carcinogenesis and apoptosis of the cell. It is expected that it may be possible to destroy cancer cells by controlling the expression of c-Myc protein. However, as described above, c-Myc protein causes apoptosis in cases of both expression level increases and decreases. Thus, it is not easy to induce apoptosis by controlling the expression of c-Myc protein. Furthermore, a method using glucocorticoid or an antisense strand of the c-myc gene as a means for inducing apoptosis by suppressing c-Myc protein expression has been proposed (Thompson, E. B., Ann. Rev. Physiol. 60: 575-600, 1998; Thulasi, R., et al., J. Biol. Chem. 268: 18306-18312, 1993; Zhou, F. et al., J. Steroid Biochem. Mol. Biol. 73:195-202, 2000; Thompson, E. A. et al., Cancer Res., 51: 5544-5550, 1991; Helmberg, A. et al., EMBO J., 14: 452-60, 1995). However, such method is not preferable for clinical use in terms of side effects and stable effects.
Therefore, an object of the present invention is to provide a novel means for stably and surely inducing cell apoptosis using the c-myc gene as a target.
Another object of the present invention is to provide a method for inducing apoptosis in cells within animal individuals, and particularly cells, the existence of which is harmful to their host animals, using the above means for inducing apoptosis.